Entry of the human immunodeficiency virus type-1 (HIV-1) into the target cell is initiated by binding of the envelope glycoprotein surface unit gp120 to CD4 and a coreceptor (CXCR4 or CCR5) followed by gp41 refolding from its native conformation to a fusion-active conformation, a six-helix bundle consisting of three pairs of the N- and C-terminal heptad repeats (NHR and CHR, respectively). This refolding process brings the viral and target cell membranes together for fusion. Peptides derived from CHR region, named C- peptides, are able to bind to the gp41 NHR region to block formation of the fusion-active gp41 core, thereby inhibiting gp41-mediated membrane fusion. We previously hypothesized that a small molecule that binds to the gp41 NHR, especially the cavity region, may block the gp41 six-helix bundle formation and inhibit HIV-1 fusion. This hypothesis was validated by identification of several non-peptidic HIV-1 fusion inhibitors with "drug-like" properties, e.g., NB-2 and NB-64, which block the fusion-active gp41 core formation. Most recently, we identified a series of small molecule compounds with structures similar to NB-2 and NB-64, but having much improved anti-HIV-1 activity. Some of these compounds, e.g., NB-154 and NB- 206 have IC50 (concentration for 50% inhibition of HIV-1 infection) values are in nanomolar range. These compounds effectively inhibited HIV-1 fusion and replication by targeting gp41 and may serve as leads for developing novel anti-HIV-1 therapeutics. We hypothesize that more potent small molecule HIV-1fusion inhibitors as anti-HIV-1 drug candidates can be designed based on the structures of NB-154 and NB- 206 by lead optimization. The specific aims of this project are: 1) to establish a focused chemical library based on the structures of the lead compounds NB-154 and NB-206; 2) To determine the structure-activity relationship (SAR) for lead optimization and for designing more potent anti-HIV-1 compounds; 3) to study the mechanism of action of the most active HIV-1 fusion inhibitors; and 4) to evaluate the efficacy of the selected compounds on in vitro infection by cell-free and cell-associated primary HIV-1 strains with distinct genotypes and phenotypes. The long-term goal is to develop novel small molecule HIV-1 fusion inhibitors asa new class of anti-HIV-1 drugs. Since more than 40 million people in the world are living with HIV/AIDS, development of new anti-HIV drugs is urgently needed for treatment of patents with HIV infection/AIDS, especially for those who fail to respond to the current antiretroviral drugs. Therefore, this research is relevant to public health.